| Intrauterine adhesion(IUA)is a common gynecological disease among women of reproductive age.The main clinical manifestations of IUA are abnormal menstruation,infertility and repeated abortion,etc.IUA often induced by intrauterine operation,which cause endometrial injury and fibrosis,but the exact pathogenesis of IUA has not been clarified.At present,the main treatment for intrauterine adhesion are hysteroscopic adhesion resection,using balloon catheter or intrauterine devices for intrauterine separation,and taking oral hormone drugs to promote endometrial regeneration.Clinical consensus believes that appropriate methods after intrauterine surgery such as injecting sodium hyaluronate or medical chitosan have certain preventive effect on intrauterine adhesion.However,the poor prognosis of moderate and severe intrauterine adhesion and the bad achievement of the desired effect by repeated operations are the main problems.The current research focuses on the promotion of endometrial regeneration and prevention of intrauterine adhesion after intrauterine operation.Hydrogels are three-dimensional cross-linked network structures formed by hydrophilic macromolecules with large amounts of water.Their soft nature similar to living organisms,modifiability by various functional groups contained,and biocompatibility by high water content make hydrogels widely applied as tissue engineering materials in drug transportation,tissue scaffolds and other aspects.Chitosan is the only alkaline polysaccharide in nature with a wide range of sources and has been used as antibacterial,antioxidant and hemostasis.In the first part of this study,chitosan was used as the main raw material to manufacture temperature sensitive hydrogels,and the properties of hydrogels were characterized.The components of the hydrogels were analyzed by Fourier transform infrared spectroscopy(FTIR),and scanning electron microscope(SEM)was employed to characterize the microstructure of the hydrogels.At the same time,in vitro degradation performance and in vitro swelling performance of the hydrogels were measured.Finally,bovine serum albumin was used as the target molecule to measure the sustained release behavior of the hydrogels.The results showed that the formation of hydrogel mainly depended on the p H response of chitosan solubility and the electrostatic interaction between chitosan and heparin.The hydrogels showed a porous network structure which can hold and transfer small molecules.Hydrogels showed certain swelling property and good degradation stability in vitro.Hydrogels also showed the same sustained release behavior with different loading amount of bovine serum albumin,indicating its feasibility as a drug sustained release carrier.In the second part of this study,the biosafety of chitosan based hydrogels was tested according to the national standard coded with GB/T 16886.The cytotoxicity of hydrogels was determined by MTT method,and transwell test was performed to check the influence of hydrogels on cell migration ability,the local subcutaneous implant test was used to detect the in vivo degradation performance of hydrogels,systemic toxicity testing was administered by intravenous injecting the leaching solution of the hydrogels,and the effects on erythrocyte sedimentation rate and blood coagulation function was also tested.The results showed that there was no significant difference in cell proliferation rate between the experimental group and the control group indicating that hydrogels showed no cytotoxicity.There was no significant difference in the migration ability between the group treated with growth factor control releasing hydrogel and the positive control group,and the sustained releasing behavior could maintain the promoting effect on the cell migration for a long time.The results of local subcutaneous test showed that the volume and weight of subcutaneous hydrogels decreased gradually after implantation,which proved that the hydrogels could degrade in vivo.The results showed that there was no significantly different in the behavior,state and body weight of the rats between experimental group and the control group.Erythrocyte sedimentation test and coagulation function test indicated that hydrogel showed no effect on erythrocyte sedimentation rate and coagulation function.The above results show that chitosan hydrogels showed no toxicity and biosafety,it can be used as bioengineering materials for further research.In the third part of the experiment,a rat model with endometrial injury was established by dual-damage method,and chitosan hydrogel with control releasing of stromal cell-derived factor-1(SDF-1α)was used to prevent the formation of intrauterine adhesion.The morphology,thickness and number of glands of rat endometrium were observed by H&E staining,the degree of endometrial fibrosis was observed by Masson trichrome staining,immunohistochemical staining was used to observe the reconstruction of the endometrial epithelium and blood vessels in the endometrium.Immunofluorescence staining was used to observe the positive expression of c-kit in the endometrium,and Western Blot was used to check the expression of c-kit,VEGF and TGF-β1 in the endometrium.Finally,the recovery of physiological function was detected.The results of H&E staining showed that the structure of endometrium recovered better in the group treated with growth factor control releasing hydrogel,and there was no significant difference in the thickness of endometrium and the number of glands compared with the control group.The results of Masson trichrome staining showed that the degree of endometrial fibrosis in the group treated with growth factor control releasing hydrogel was significantly lower than that in the model group,and there was no significant difference between the group treated with growth factor control releasing hydrogel and the control group.The results of immunohistochemical staining showed that the recovery of endometrial epithelium in the group treated with growth factor control releasing hydrogel was well and the vascular reconstruction in the endometrium was completed,which showed no significant difference from the control group.The results of immunofluorescence staining showed that the group treated with growth factor control releasing hydrogel recruited a number of c-kit positive cells in the injured endometrium for a long time,which is significantly more than the other treatment groups.Western Blot results showed that the expression of c-kit in the endometrium of the group treated with growth factor control releasing hydrogel was higher than other experimental groups at Day 4 after treatment,and the expression of VEGF and TGF-β1 was lower than other experimental groups.At Day 7 after treatment,the expression of c-kit was significantly higher than other experimental groups,and the expression of VEGF and TGF-β1 was lower than other experimental groups,and there was no significant difference between the group treated with growth factor control releasing hydrogel and the control group.The physiological functional testing results showed that the uterine function of the group treated with growth factor control releasing hydrogel recovered well,and there was no significant difference between the group treated with growth factor control releasing hydrogel and the control group.The results indicated that hydrogels with controlled releasing SDF-1α could effectively prevent the formation of intrauterine adhesion.Therefore,the chitosan based temperature sensitive hydrogels with sustained release behavior showed biological safety,could be used to prevent the formation of intrauterine adhesion with SDF-1α loaded,which provides a good reference for clinical surgery. |
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